EP3956461A1 - Methanation method in a bioreactor under continuous cell-retention conditions - Google Patents
Methanation method in a bioreactor under continuous cell-retention conditionsInfo
- Publication number
- EP3956461A1 EP3956461A1 EP20722498.1A EP20722498A EP3956461A1 EP 3956461 A1 EP3956461 A1 EP 3956461A1 EP 20722498 A EP20722498 A EP 20722498A EP 3956461 A1 EP3956461 A1 EP 3956461A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- culture medium
- phase
- cell retention
- cell
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 74
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims abstract description 216
- 230000014759 maintenance of location Effects 0.000 claims abstract description 178
- 230000000696 methanogenic effect Effects 0.000 claims abstract description 85
- 244000005700 microbiome Species 0.000 claims abstract description 83
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 75
- 230000002503 metabolic effect Effects 0.000 claims abstract description 45
- 238000012258 culturing Methods 0.000 claims abstract description 38
- 238000010924 continuous production Methods 0.000 claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 claims description 103
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 67
- 239000001963 growth medium Substances 0.000 claims description 56
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 37
- 239000011707 mineral Substances 0.000 claims description 37
- 229910052757 nitrogen Inorganic materials 0.000 claims description 33
- 230000010261 cell growth Effects 0.000 claims description 28
- 239000012528 membrane Substances 0.000 claims description 26
- 239000007789 gas Substances 0.000 claims description 23
- 230000002829 reductive effect Effects 0.000 claims description 22
- 241000203069 Archaea Species 0.000 claims description 13
- 230000001276 controlling effect Effects 0.000 claims description 11
- 230000001105 regulatory effect Effects 0.000 claims description 11
- 241001302035 Methanothermobacter Species 0.000 claims description 9
- 230000032823 cell division Effects 0.000 claims description 9
- 238000001223 reverse osmosis Methods 0.000 claims description 9
- 229910017974 NH40H Inorganic materials 0.000 claims description 8
- 238000001704 evaporation Methods 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 238000004064 recycling Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- 239000011148 porous material Substances 0.000 claims description 7
- 229910052979 sodium sulfide Inorganic materials 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 claims description 3
- 150000003868 ammonium compounds Chemical group 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 2
- 241000202974 Methanobacterium Species 0.000 claims description 2
- 241000202987 Methanobrevibacter Species 0.000 claims description 2
- 241000203353 Methanococcus Species 0.000 claims description 2
- 241000204675 Methanopyrus Species 0.000 claims description 2
- 241000205276 Methanosarcina Species 0.000 claims description 2
- 238000001728 nano-filtration Methods 0.000 claims description 2
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 23
- 239000006143 cell culture medium Substances 0.000 abstract description 14
- 210000004027 cell Anatomy 0.000 description 235
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical class N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 103
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 52
- 238000002474 experimental method Methods 0.000 description 52
- 229910021529 ammonia Inorganic materials 0.000 description 47
- 229910002092 carbon dioxide Inorganic materials 0.000 description 47
- 238000006243 chemical reaction Methods 0.000 description 39
- 230000009467 reduction Effects 0.000 description 32
- 235000015097 nutrients Nutrition 0.000 description 28
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
- 230000003698 anagen phase Effects 0.000 description 18
- 239000002609 medium Substances 0.000 description 17
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 15
- 229910052739 hydrogen Inorganic materials 0.000 description 15
- 239000001257 hydrogen Substances 0.000 description 15
- 238000012360 testing method Methods 0.000 description 15
- 238000011084 recovery Methods 0.000 description 14
- 239000002028 Biomass Substances 0.000 description 9
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- 230000012010 growth Effects 0.000 description 6
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 239000000919 ceramic Substances 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical class [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- -1 therefore Chemical compound 0.000 description 4
- 240000002900 Arthrospira platensis Species 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000001651 autotrophic effect Effects 0.000 description 3
- 239000011942 biocatalyst Substances 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- VDQVEACBQKUUSU-UHFFFAOYSA-M disodium;sulfanide Chemical compound [Na+].[Na+].[SH-] VDQVEACBQKUUSU-UHFFFAOYSA-M 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
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- 238000001471 micro-filtration Methods 0.000 description 3
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 3
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 235000016425 Arthrospira platensis Nutrition 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229940011019 arthrospira platensis Drugs 0.000 description 2
- 238000010923 batch production Methods 0.000 description 2
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- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000002803 fossil fuel Substances 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
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- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 230000007306 turnover Effects 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- 241001464430 Cyanobacterium Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000197200 Gallinago media Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241001302042 Methanothermobacter thermautotrophicus Species 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 229910003424 Na2SeO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
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- 150000001413 amino acids Chemical class 0.000 description 1
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- 238000013459 approach Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
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- 239000011575 calcium Substances 0.000 description 1
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- 150000001721 carbon Chemical group 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
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- 229910010293 ceramic material Inorganic materials 0.000 description 1
- 150000003841 chloride salts Chemical class 0.000 description 1
- 239000003245 coal Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
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- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
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- 229910052750 molybdenum Inorganic materials 0.000 description 1
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
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- 229910052759 nickel Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
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- 239000012466 permeate Substances 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
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- 239000012465 retentate Substances 0.000 description 1
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- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
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- 235000013343 vitamin Nutrition 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/023—Methane
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/107—Apparatus for enzymology or microbiology with means for collecting fermentation gases, e.g. methane
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/02—Atmosphere, e.g. low oxygen conditions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2511/00—Cells for large scale production
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Definitions
- the present invention refers to a highly efficient method for producing biogenic methane using H 2 and CO 2 by methanogenic microorganisms in a bioreactor even under conditions of reduced nitrogen supply in the methane production phase.
- Methane has the highest energy density per carbon atom among fossil fuels and its potential for energy conversion is far greater than any other natural gas, obtained directly by combustion in presence of oxygen or using fuel cells to produce electricity. Methane’s potential for energy generation has become increasingly relevant in the global market.
- methane constitutes a sustainable and renewable energy source and already today increasingly substitutes coal and other fossil fuels.
- methanogenic microorganisms For industrial production of methane using Archaea, e.g., Methanothermobacter thermautotrophicus strain UC 120910 (ECH100 or ECHOIOO). - deposited and commercially available may regularly be used.
- Archaea e.g., Methanothermobacter thermautotrophicus strain UC 120910 (ECH100 or ECHOIOO). - deposited and commercially available may regularly be used.
- a culture of hydrogen using methanogenic microorganisms catalyses the methanation reaction as follows:
- the water produced by this methanation process (see equation 1), which is also called “metabolic water” or“free water” has to be continuously discharged during the methanation process in a sewage system to to maintain a constant liquid level in the bioreactor and to prevent overflowing of the bioreactor due to the increase in the liquid volume.
- This discharged water also medium components like minerals / nutrients (salts, ions, micronutrients) important for maintaining the methanogenic microorganisms and to allow for effective methanation are lost.
- Another problem associated with the production of metabolic water is the dilution of the medium components within the culture medium.
- RO Reverse Osmosis
- a method to convert H 2 and CO 2 into methane by methanogenic microorganisms in a bioreactor in the methane production phase comprising the steps: i. culturing the methanogenic microorganisms in a suitable liquid culture medium comprising minerals in a continuous process; ii. culturing the methanogenic microorganisms under cell retention conditions; iii. contacting the methanogenic microorganisms with at least one feeding gas comprising CO 2 and H 2 ; iv. continuously removing metabolic water in the culture medium from the bioreactor; v. collecting methane or a methane enriched gas composition.
- the method of the present invention does comprise a step of culturing methanogenic archaea, which is based on typical culture conditions for archaea, which have been previously described and which are known to the practitioner. Such conditions are influenced and controlled - according to the skills of a practitioner by common parameters affecting the culture including temperature, pressure, volume, humidity, salt content, conductivity, carbon content, nitrogen content, vitamin content, amino acid content, mineral content, or any combination thereof.
- the step of culturing the methanogenic microorganism in the method to produce methane from CO 2 and H 2 containing gas or gases in a bioreactor comprises: keeping said methanogenic microorganism in a suitable liquid culture medium providing suitable minerals or nutrients such as e.g. a nitrogen source and salts.
- CO 2 and H 2 may be e.g. applied as pure gases.
- CO 2 may be also or alternatively delivered using the supply of industrial gases.
- Such industrial gases depending on their source may comprise very different gas compositions. They have primarily in common that they contain a relatively high amount of CO 2 in comparison to air. They may contain a normal (air-like) partial amount of oxygen and/or nitrogen, however depending on their origin they may also be oxygen free.
- they may contain substantial amounts of at least one of the following, particularly carbon monoxide, hydrogen and hydrogen sulfide, other sulphur compounds (sulfides, disulfides, thiols), siloxanes (organic silicon compounds), halogenated compounds, ammonia, and organochlorines, i.e. pesticides and other synthetic organic compounds with chlorinated aromatic molecules.
- the inventors of the present invention have advantageously and surprisingly found by running a bioreactor under cell retention condition that this condition increases the overall efficiency of the system as the feeding with CO 2 and H 2 is essentially used for methane production.
- the efficacy of the system was observed to be 30% or higher or preferably to be 50 % or higher than in comparable experiments where no cell retention conditions were applied. Processes, which are included in the calculation of this efficacy are the reduction of costs, saving of nutrients while increasing the overall methanation rate.
- a "phase" in the sense of the invention describes a condition or state of the methanogenic microorganisms in the bioreactor of the invention, which is characterized by specific fermentation conditions, which are applied to the methanogenic microorganism, e.g., the ratio of the partial pressures of hydrogen and carbon dioxide or a specific value or range of at least one nutrient, which is applied, e.g. ammonium and/or the settings of the bioreactor to keep cells in the reactor (cell retention) or not.
- a “cell growth phase” according to the present invention is a phase mainly characterized by an increase of the biomass of the methanogenic microorganisms by cell division and cell growth.
- a “methane production phase” according to the present invention is a phase mainly characterized by methane production rather than cell division and cell growth.
- the cells may also or may not produce methane and during any methane production phase, the overall biomass may also increase.
- cell retention conditions refers to conditions in a running bioreactor, which enable and guarantee that cells, i.e. the methanogenic
- microorganisms are kept inside the bioreactor or are recycled and reintroduced into the bioreactor. Many ways to enable such cell retention conditions in a running bioreactor in a continuous process are possible and easily accessible for a skilled person.
- the growth phase and/or the methanation production phase may be performed via culturing the cells under cell retention conditions. It is also possible that a phase under cell retention conditions according to the present invention is flanked alternatively one side or both sides by a growth phase or a methanation production phase performed via culturing the cells under no cell retention conditions.
- culturing the cells under no cell retention conditions is meant a situation in a running bioreactor, which does not enable and does not guarantee that cells, i.e. the methanogenic microorganisms are kept inside the bioreactor, i.e. methanogenic microorganisms will be washed out of the bioreactor during this phase.
- methanation or methanogenesis or biomethanation, is understood as the production of methane or a methane enriched gas composition as carried out by methanogenic microorganisms, such as those included in a list of methanogenic
- the methanation reaction as previously known and as suitable according to the present invention, consumes H 2 and CO 2 at a stoichiometry of 4:1 (see above, equation 1).
- methanogenic microorganisms are cultured in a bioreactor in order to produce biomethane.
- Such methanogenic microorganisms, or autotrophic methanogenic microorganisms may be anaerobic archaea or even recently classified aerotolerant archaea, either in pure strains, or in consortia with a plurality of, i.e. two or more, strains, or in mixed cultures wherein methanation may be also encouraged by syntrophic exchange across different species.
- a“bioreactor” stands for a reactor, and is either a bioreaction vessel, or a bioreaction enclosure, or a bioreaction tank, and/or at least a bioreaction chamber, and/or a cell, or a combination thereof, as also intended in the state of the art, able to withstand variations of e.g. temperature and/or pressure, among others, and/or able to maintain whichever imparted values of e.g. temperature, and/or pressure are assigned or have to be maintained, before, after or during the reaction process, and wherein the intended reactions relevant for carrying out the invention may take place.
- Such reactions are understood as bioreactions as they pertain to the domain of reactions wherein microorganisms are involved, and herein referring to their normal physiology - such as e.g. metabolic fermentation, or aerobic or anaerobic digestion - and that, as such, require suitable environments, suitable cultures of microorganisms, suitable culture mediums and suitable reactants to occur.
- a bioreactor in the meaning of the invention performs reliably within the tolerance values of each variable in order to enable the method as disclosed, and it is expected to allow the listed steps to be carried out reliably over time.
- a suitable reactor for culturing methanogenic microorganisms may be, by means of example only, a shake tank bioreactor, a continuous stirred tank bioreactor, an intermittent stirred tank bioreactor, a hollow fiber membrane bioreactor, a bubble column bioreactor, an internal-loop airlift bioreactor, an external-loop airlift bioreactor, a fluidized bed bioreactors, a packed bed bioreactor, a photo-bioreactor, a trickle bed reactor, a microbial electrolysis cell, etc., and/or combinations thereof.
- a reactor may be chosen that most closely addresses the specific dynamics of a culture or the convenience by which methane is hereby extracted.
- a bubble column reactor or a variant of it, such as an airlift bioreactor, or a continuously stirred tank reactor, and/or any of the above, may be used to conveniently carry out the method as described and a continuous culture is preferred, wherein near-balanced growth, with little fluctuation of nutrients, metabolites, cell number and biomass are observed.
- the method herein disclosed is concerned with the culturing of methanogenic microorganisms in a “continuous process”, wherein such continuity is understood as continuity in the production of methane and continuity in the culture, wherein no step of separating inactive terminal biomass from active members of the colony is required. It is instead encouraged that dead biomaterial is kept in the reactor together with the active members across several stages of growth, as it is found advantageous that said biomass or biomaterial provide further substrate for the active culture, intensifying nutrition availability.
- a continuous supply of suitable reactants e.g.
- methane is produced by methanogenic archaea from single strains or in mixed cultures, wherein a mixed culture is either a culture where a plurality of, therefore two or more, strains may also be employed, or a culture where a plurality of additional species interact with methanogenic archaea, or any combination thereof.
- “Metabolic water” refers to water or H 2 0 molecules, which are produced by the methanogenic organisms during metabolic activity and the process of methanogenesis, i.e. mainly in the methane production phase.
- step iv. the removing of the metabolic water in the culture medium from the bioreactor is done
- step i. comprises at least one cycle of culturing the methanogenic microorganisms under: a first phase in a continuous process in a suitable liquid minerals containing culture medium comprising a reduced supply of at least one mineral; followed by a second phase, characterized by refreshing the culture medium; optional followed by a third phase in a continuous process comprising a reduced supply of at least one mineral.
- A“at least one mineral” according to the present condition within the first phase and/or third phase refers to typical minerals, which are present in classical cell culture mediums, e.g. a nitrogen source and/or salts.
- the“at least one mineral” is a nitrogen source.
- the“at least one mineral” is a salt, e.g. a chloride containing salt.
- the chloride can be present in the salt respectively dissolved as saline solution as the anion of NaCl, MgCl, KCl, NH4CI or any other suitable chloride salt known to the skilled person.
- The“at least one mineral” which supply is decreased may be the same or be a different one in the first and the third phase.
- A“refreshing of the culture medium” according to the present invention within the second phase can be realized by changing the cell culture medium at least partly or by adding at least one nutrient, which triggers cell division and cell growth.
- Nutrients, which trigger cell growth and cell division are well known by an artisan and include the addition or the increase of a nitrogen source, a sulfur source, phosphorous and cell growth factors.
- a combination of the described options for refreshing of the culture medium is also a possible option according to the present invention.
- This second phase can optionally be followed by a third phase, wherein the cells are again cultured in a continuous process comprising a reduced supply of at least one mineral. Then, the second phase is a transition phase flanked between two phases in a continuous process within the at least one cycle.
- Such a “refreshing of the culture medium” may be but not necessarily be applied every month, every half year for at least one day or at least one day to five days or at least one day to four days at least one day to three days.
- additional nutrients are supplied to the cell culture medium continuously depending on the need of the cultured cells and the consumption of nutrients by the cells in a continuous process.
- step ii. comprises at least one cycle of culturing the methanogenic microorganisms under: a fourth phase under cell retention conditions; followed by
- a fifth phase characterized by culturing the cells under no cell retention
- A“culturing the cells under no cell retention conditions” according to the present invention within the fifth phase refers to conditions in a running bioreactor, which does not enable and does not guarantee that cells, i.e. the methanogenic microorganisms are kept inside the bioreactor, i.e. methanogenic microorganisms will be washed out of the bioreactor during this phase.
- This fifth phase can optionally be followed by a sixth phase, wherein the cells are again cultured under cell retention conditions. Then, the fifth phase is a transition phase between two phases under cell retention conditions within the at least one cycle.
- the inventors of the present invention have found, that culturing the methanogenic
- microorganisms under such no cell retention conditions may be advantageous at a certain running time of the reactor.
- This phase under such no cell retention conditions may promote cell division and cell growth, which may have a positive effect on the overall methanation process efficiency.
- Such no retention conditions may be but not necessarily be applied every month, every half year for at least one day or at least one day to five days or at least one day to four days at least one day to three days.
- the step of culturing the methanogenic organisms comprises: controlling and reducing the supply of a nitrogen source in the methane production phase to receive a nitrogen source concentration in the culture medium in an amount of 0.2 moL/L/day to 0 moL/L/day or of 0.02 moL/L/day to 0.005 moL/L/day preferably between 0.11 moL/L/day 0.005 moL/L/day.
- Methanogenic microorganisms generally need a nitrogen source and accordingly all published prior art documents the supply of nitrogen in one or the other way. Nevertheless, the inventors of the present invention have surprisingly found, that by cultivating methanogenic microorganisms under cell retention conditions according to the present invention it is possible to tremendously reduce or even completely stop the supply of the nitrogen source in the methane production phase and still enable a high and quite stabilized methanation rate while observing a stabilized maintained cell culture number.
- a phase of culturing the cells under cell retention conditions flanked by two other phases, e.g. two cell growth phases under no cell retention conditions. It is believed - without being bound by that theory - that when all methanogenic microorganisms are kept inside the reactor, growth of the cells is only required in a pronounced amount in the“growth phase” at the beginning of the start-up of a reactor and not during methane production phase, resulting in a nitrogen savings for the cells during the methane production phase. However, if a cell population of sufficient number is directly applied in the start-up of the reactor a growth phase is not necessary.
- the reason why the total cell number of the methanogenic microorganisms stays stabilized over time even under prolonged reduction or even stop of the external supply of the nitrogen source in the methane production phase is that the nitrogen during natural turn-over of pre-existing cell mass of the methanogenic microorganisms developed in the growth phase is used to build up new cells during the archaea generation cycle in the methane production phase. This would mean that the nutrients of e.g. dying methanogenic microorganisms including nitrogen are recycled by the living methanogenic microorganisms to grow and/or to build up new cells by division.
- the present invention is besides others characterized by a step of controlling the external supply of the nitrogen source and/or the (resulted) concentration of the nitrogen source (i.e. ammonia) within the cell culture medium.
- controlling is understood in the general common meaning of keeping under constant monitoring the parameters related to the culture and essentially measuring said parameters or status indicators, using common methodologies and measuring instrumentation known in the art, since it might not be sufficient to keep under constant monitoring and therefore only control this parameter of the culture; therefore a further embodiment of the present invention comprises in particular regulating the nitrogen source concentration within the cell culture medium continuously.
- regulating is intended as actively maintaining a“given value” or a given value span for a parameter, e.g. the nitrogen source concentration of the culture, by using appropriate means to do so.
- a “given value” according to the invention may be a defined value with given tolerances, tolerances within the measurements system or tolerances due to the variability within the culture or due to the culture diversity, wherein said value is suitable for enabling methanation; or a given value may be a range of suitable values, which achieve the same effect on methanation as a given value.
- methanogenic organisms may include common inorganic elements, in their elemental forms or in any suitable non-toxic salts thereof, e.g. sodium, potassium, magnesium, calcium, iron, chloride, sources of sulfur, e.g. hydrogen sulfide or elemental sulfur, phosphorus sources, e.g. phosphate, nitrogen sources, e.g. ammonium, nitrate or nitrogen gas.
- Typical salts utilized for culturing methanogenic organisms according to the present invention are NaCl, KH 2 P0 4 , FeCl2-4H 2 O, Na 2 SeO 3 , Na 2 S, NH 4 OH and MgCl 2 .
- methanogenic organisms further comprise: providing a sulfide source, preferably in the form of Na 2 S in the
- the step of culturing the methanogenic microorganism in the method to produce methane from industrial gases containing CO 2 in a bioreactor further comprises: providing a sulfide source, preferably in the form of Na 2 S in the culture medium; keeping the culture conditions facultatively anaerobic and/or anaerobic; optionally stirring the culture, wherein the stirring of the culture can be carried out regularly, in intervals, continuously, or keeping the soluble culture at least in a certain slow and constant movement; removing metabolic water from the culture continuously; and keeping the temperatures in a range between 32°C and 80°C; preferably 50-70°C or around 62°C.
- temperatures may vary according to the presence of selected microorganism species within the culture, each of which better thrive within set ranges of temperatures, for most of the methanogenic microorganisms increased temperatures are not detrimental, and they may even assist in optimizing cellular metabolism and thus metabolic turnover or even methanation.
- a temperature must be controlled by energetic regulation; in this regard it is to be considered a valuable feature to reduce energy expenditure by enabling temperature control.
- the method of the present invention was found to be most efficient in a temperature range between 32°C and 85°C, or alternatively 50 to 70°C or further alternatively around 62°C at atmospheric pressure. If according to some embodiments one or more steps of the method according to the invention are carried out in a pressurized atmosphere, then the pressure is chosen to be preferably up to 16 bar, alternatively up to 20 bar, alternatively up to 50 bar, alternatively up to 68 bar, alternatively up to 110 bar or even up to 420 bar.
- the present invention also refers to a culturing process at pressures equal or between the range of 1 to 10 bar.
- High pressure e.g. 16 bar, 20 bar, 35 bar, 40 bar or 60 bar and correspondingly, higher temperatures, which would allow the same hydrogen solubility as at a temperature range between 32°C and 85°C, or alternatively 50 to 70°C or further alternatively around 62°C at atmospheric pressure are also encompassed.
- Methanogenic microorganisms in general, may live and grow also in a plurality of other and even extreme temperature ranges up to and well above 100°C, e.g. 140°C; accordingly, the above temperature range is an indication of a preferred range, but it is not to be understood as limiting the scope of the invention.
- the culturing of the methanogenic organisms comprises a cell growth phase prior to the methane production phase, comprising the steps of: controlling and regulating the concentration of a nitrogen source within the culture medium in a range of 0.2 moL/L/day to 0.005 moL/L/day, preferably of 0.02 moL/L/day to
- microorganisms culturing the methanogenic microorganisms up to a density in
- the culture medium measured as OD610 being at least 1,9 up to 200 or at least 20 up to 120, preferably at least 60 up to 100 and corresponding to a dry weight of the
- the ODeio optical density at 610 nm
- the ODeio optical density at 610 nm or briefly optical density of microorganisms in a culture is a viable parameter to measure the cell count or concentration at each time point.
- a straightforward relationship between a given cell count and the efficiency of the microorganisms in a culture it does not appear to have been universally established, nevertheless in the understanding of the results of the method according to the present invention, a high density culture produces advantageous results in terms of methane production and yield.
- the optical density (OD) of the culture according to the present invention is measured utilizing common methods and standards known in the art.
- Optical density, or, rather, turbidity measurements as a form of cell counting are performed using a spectrophotometer, is typically operated around or at 600 nm, but accordingly other wavelengths may be suitable.
- the optical density may vary according to the measurement setup, it is often useful to indicate the dry weight or biomass density of the microorganisms in the culture as a measure of the amount of cells present in a culture at a given time point or growth phase. It is possible to establish a correlation between measurements of OD of a given culture at a given growth stage and dry weight by building a curve of a number of different OD values of the culture obtained at different concentrations and measuring the dry weight of the dried sample of culture accordingly, using standard methods known in the art. This will provide a set of data point of dry weight as a function of the optical density; the slope of the regression line of such data set usually defines the correlation between dry weight and optical density.
- the culture of the methanogenic microorganisms can be guided or led into a high density culture with an OD 610 of at least 14, but preferably above 20, further also above 30, further above 40 and even up to 120 or 200 by supplying sufficient nutrient to the culture and simultaneously removing free or metabolic water from the culture.
- the method of the present invention can thus be suitably performed in culture of one or more strains of methanogenic microorganism, having throughout the various developmental stages a measurable between 60 - 200; further an OD 610 between 14 - 120; further an OD 610 between 20 - 120; further an OD 610 between 30 - 120; further an OD 610 between 40 - 120; further an OD 610 between 50 - 120; further an OD 610 between 50 - 100; further an OD 610 between 14 - 80; further an OD 610 between 20 - 80; further an OD 610 between 30 - 80; further an OD 610 between 40 - 80; further an between 20 - 80; further an between 30 - 40; further an between 40 - 60; further an between 20 - 40.
- a high optical density corresponding to a high number of cells is obtained into the growth phase and maintained by keeping the members of the culture in the bioreactor across the entire stages of their lives to their terminal stage, so that the remains of the inactive cellular bodies may provide nutrients to the active members of the culture.
- the nitrogen source is but not limited to ammonium compounds, preferably in the form of NH 4 0H or NH 4 Cl or combinations of the aforementioned.
- the nitrogen source is an ammonium compound, preferably in the form of NH 4 0H.
- the method further comprises the step of setting an initial pH value to be at a given value of below pH 9, below pH 8 or at pH 7 and subsequent continuously controlling the pH value.
- the inventors of the present invention have surprisingly found that after setting an initial pH to be at a given value, this pH value can be maintained over the whole experimentations throughout the various conditions of the present invention.
- this pH value can be maintained over the whole experimentations throughout the various conditions of the present invention.
- Unexpectedly was that even after running the bioreactor under successive phases of reduction (up to 440 h) and complete stopping of the supply of the nitrogen source (up to 216 h), e.g. concretely in the form of NH40H without adding further amounts of a base as surrogate for the missing basic OH moiety of the NH40H compound, this does not require an additional supply of a base (and/or acid) to maintain the pH at a given value.
- providing the advantage to reduce the process cost for running the bioreactor as the supply of the nitrogen source are reduced while saving time and costs to continuously control and regulate the pH at a given value.
- the pH is optionally continuously controlled and/or alternatively further regulated, i.e. stabilized to be kept at a given different value.
- the step of controlling and regulating the pH value continuously to be kept at a given different value is done by dosing suitable amounts of a base and/or an acid, e.g. NaOH/HCl or NH 4 0H/HCI to the culture.
- the removing of the metabolic water comprises the step of filtrating excess water away from the culture medium and/or comprises the step of evaporating excess water from the culture medium.
- Examples 1 -3 The means how to evaporate excess water from the culture medium in a running bioreactor is well known by a skilled person.
- One not limiting way how this can be performed is disclosed in industrial scale - see Example 5.
- the step of filtrating excess water away from the culture medium is performed by reverse osmosis using at least one semipermeable membrane for water in contact with the culture medium.
- the technique and how to perform reverse osmosis are well known to the skilled person. One not limiting way how this can be performed is disclosed in industrial scale - see Example 4.
- the at least one membrane semi-permeable for water in contact with the culture medium is located in the proximity of a device, e.g. a tube with is in contact with the culture medium and is under a negative pressure resulting in a net efflux of water from the bioreactor.
- the step of filtrating excess water away comprises the step of: removing fractions of cell-free culture medium from the bioreactor by filtration through at least one porous membrane in contact with the culture medium, preferably having a pore size of 0.4 to 0.1 mm, particularly preferably of 0.3 mm; and optionally, subsequently concentrating the minerals from the removed culture medium preferably by at least one further filtration step, e.g. nanofiltration, ultrafiltration and/or by at least one distillation step; and optionally, at least partially recycling the concentrated minerals back to the bioreactor.
- at least one further filtration step e.g. nanofiltration, ultrafiltration and/or by at least one distillation step
- filtration means of appropriate pore-diameter all cells are kept inside the reactor (cell retention) and only the produced process water with the soluble components of the cell culture medium (cell-free fractions of cell culture medium) are removed from the bioreactor.
- the porous membrane in contact with the culture medium suitable for microfiltration may be located anywhere in the bioreactor as long as a continuous flow of the removed fractions of cell-free culture medium is possible.
- the membrane can e.g. be located close to the surface of the cell culture medium in the direction of the top of the bioreactor or be directed close to or at the bottom of the bioreactor.
- An example is depicted in Fig. 14.
- a laboratory scale reactor was supplied with H 2 , generated by an electrolyser, and CO 2 .
- the flow rates of hydrogen and carbon dioxide were adjusted typically to a 4:1 ratio.
- the produced metabolic water of the reactor was removed with a ceramic filter, which was located inside the reactor. With this membrane the complete metabolic water containing dissolved nutrients was removed, only cells were retained in the reactor. To balance the loss of nutrients, media stock solutions were dosed according to the discharge volume.
- all minerals of the formerly removed fractions of cell-free culture medium are fully recovered and the concentrated minerals are recycled back to the bioreactor.
- all minerals of the formerly removed fractions of cell-free culture medium are at least partially recovered and the concentrated minerals are recycled back to the bioreactor.
- Non-limiting examples of at least partially or fully recovered minerals are Nickel, Cobalt, Iron, Potassium, or Phosphorus.
- the porous membrane is made of ceramic material, polyethylene or stainless steel.
- cells may be at least partly and only time-wise removed from the bioreactor and returned to the reactor after passing an appropriate porous membrane.
- the method further comprises the step of controlling and optionally regulating the concentration of at least one entity of the minerals in the culture medium by additional adding of minerals.
- media stock solutions can be dosed according to the discharge volume.
- the step of additional adding to the culture minerals or nutrients may be performed in a continuous or a discontinuous mode and includes minerals as e.g. Sodium, Tungsten,
- Methanogenic microorganism in general, may live and grow also in the presence of multiple minerals or nutrients.
- Autotrophic methanogenic microorganisms are herein intended as microorganisms which derive nutrition from inorganic reactions with their surrounding environment, e.g. by reducing carbon dioxide, to perform biosynthesis of methane.
- An example of autotrophic microorganisms is given by hydrogenotrophic microorganisms, which derive their nutrition from utilizing hydrogen; in particular, hydrogenotrophic methanogenic microorganisms are able to convert hydrogen and carbon dioxide into methane as part of their metabolic processes.
- the role of methanogenic microorganisms in the ecosystem is unique as it helps removing excess carbon dioxide and fermentation products in the final stage of decay of organic matter. In absence of methanogenesis large amounts of carbon bound to compounds from decaying matter would accumulate in anaerobic environments.
- the at least one methanogenic microorganism is selected from the group of Archaea or archaebacteria comprising of Methanobacterium, Methanobrevibacter, Methanothermobacter, Methanococcus, Methanosarcina, Methanopyrus or mixtures thereof.
- the inventors performed especially the test methanogenic microorganism Methanothermobacter thermautrophicus UC 120910 (ECHOIOO) showed such remarkably change in cell morphology during the various phases under cell retention conditions towards comparable conditions under no-cell retention conditions (see Fig. 17 A, B). There are preliminary hints that these morphology changes are reversible (data not shown).
- Fig.l Filtrating excess metabolic water away by using porous membrane filter under cell retention conditions and regulating the pH under ammonium reduction conditions (preliminary experimentation, cell retention experiment 1).
- Phases/conditions (horizontal coordinate): run time [h].
- Vertical coordinate A: OD610.
- B WD [L/L/d].
- C Conversion [%].
- Phases/conditions a: cell growth, b: cell retention (filter testing), c: methane production under no cell retention, d: production under cell retention, e: methane production under cell retention and ammonia reduction, f: methane production with cell retention without ammonia feeding.
- Fig.2 Filtrating excess metabolic water away by using porous membrane filter under cell retention conditions and regulating the pH under ammonium reduction conditions (preliminary experimentation).
- Cell retention experiment 1. Phases/conditions (horizontal coordinate): a.: cell growth, b.: cell retention filter test, c.: production without cell retention, d.: production with cell retention, e.: production with cell retention and ammonia reduction, f.: production with cell retention without ammonia feeding.
- Fig.3 Filtrating excess metabolic water away by using porous membrane filter under cell retention conditions and regulating the pH under ammonium reduction conditions (preliminary experimentation).
- Cell retention experiment 1. Horizontal coordinate: run time [h]. Vertical coordinate: D: C02 flow [L/min], E: feeding NH3 [g/l/d]. F: feeding NaOH [M/l/d]. G: NH4+ concentration supernatant [g/L], Phases/conditions: a: cell growth, b: cell retention filter test, c: production without cell retention, d: production under cell retention, e: production under cell retention and ammonia reduction, f: production under cell retention without ammonia feeding.
- Fig.4 Filtrating excess metabolic water away under cell retention conditions by using porous membrane filter with no need to regulate an initial set pH value under ammonium reduction conditions (cell retention experiment 2).
- Horizontal coordinate run time [h].
- Vertical coordinate A: OD610.
- B WD [L/L/d].
- C Conversion [%].
- Phases/condition a: cell growth, b: methane production under no cell retention, c: transition 1 and 2.
- d methane production under cell retention, e: methane production under cell retention and ammonia reduction.
- Fig.5 Filtrating excess metabolic water away under cell retention conditions by using porous membrane filter with no need to regulate an initial set pH value under ammonium reduction conditions (cell retention experiment 2).
- Phases/conditions horizontal coordinate: a: cell growth, b: methane production without cell retention, c: transition 1 and 2.
- d methane production under cell retention, e: methane production under cell retention and ammonia reduction.
- Vertical coordinate Means and standard deviation A: OD610.
- B VVD [L/L/d].
- C VVD [L/L/d].
- Fig.6 Filtrating excess metabolic water away under cell retention conditions by using porous membrane filter with no need to regulate an initial set pH value under ammonium reduction conditions (cell retention experiment 2).
- d methane production under cell retention, e:
- Fig.7 Methane production under cell retention conditions and culture medium component recycling using filters to remove excess metabolic water away (cell retention experiment s).
- Horizontal coordinate run time [h].
- Vertical coordinate A: OD610.
- B VVD [L/L/d].
- C Conversion [%].
- Phases/conditions a. cell growth under cell retention, b. methane production under cell retention, c. methane production under cell retention and nutrient recovery, d. methane production under cell retention and ammonia reduction, e. methane production under cell retention without ammonia feeding.
- Fig.8 Methane production under cell retention conditions and culture medium component recycling using filters to remove excess metabolic water away (cell retention experiment s).
- Phases/conditions (horizontal coordinate) a.
- Fig.9 Methane production under cell retention conditions and culture medium component recycling using filters to remove excess metabolic water away (cell retention experiment s).
- Horizontal coordinate run time [h].
- Vertical coordinate D: C02 flow [L/min].
- E NH3 feeding related to standard feeding [%/100] showing the reduction to 50 %, 25 % and 0 % of standard feeding.
- Phases/conditions a. cell growth under cell retention, b. methane production under cell retention, c. methane production under cell retention and nutrient recovery, d. methane production under cell retention and ammonia reduction, e. methane production under cell retention without ammonia feeding.
- Fig.10 Filtrating excess metabolic water away under cell retention conditions by using reverse osmosis filters (cell retention experiment 4).
- Horizontal coordinate run time [h].
- Vertical coordinate A: OD610.
- C Conversion [%].
- Phases/conditions a: methane production without cell retention and medium recovery, b: methane production under cell retention and medium recovery.
- Fig.ll Removal of excess metabolic water by evaporation under cell retention conditions (cell retention experiment s). Operation of an industrial scale reactor containing
- Methanothermobacter thermautrophicus UC 120910 (ECHOIOO) over a 5-day period.
- Reactor mass is the weight of the liquid medium and the biomass in the reactor, which was determined with a scale that was tared for the weight of the reactor itself.
- Fig.12 Removal of excess metabolic water by evaporation under cell retention conditions (cell retention experiment s). Operation of an industrial scale reactor containing
- Methanothermobacter thermautrophicus UC 120910 (ECHOIOO) over a 5-day period.
- Fig.13 Removal of excess metabolic water by evaporation under cell retention conditions (cell retention experiment s). Operation of an industrial scale reactor containing
- Methanothermobacter thermautrophicus UC 120910 (ECHOIOO) over a 5-day period.
- the graph shows (A.) reactor mass (kg), (B.) OD, (C.) the flow rate of biogas (Nm3/h) into the reactor.
- Reactor mass is the weight of the liquid medium and the biomass in the reactor, which was determined with a scale that was tared for the weight of the reactor itself.
- Fig.14 Reactor set up for removal of excess metabolic water by using a porous filter within the bioreactor cell culture medium (e.g., cell retention experiments 1 and 2).
- a laboratory scale reactor was supplied with H 2 , generated by an electrolyser, and CO 2 . The flow rates of hydrogen and carbon dioxide were adjusted to a 4:1 ratio.
- the temperature of the culture was 62.5 °C and the methanation reaction occurred at atmospheric pressure.
- the produced metabolic water of the reactor was removed with a ceramic filter which was located inside the reactor. With this membrane the complete metabolic water containing dissolved nutrients was removed, only cells were retained in the reactor. To balance the loss of nutrients, media stock solutions were dosed according to the discharge volume.
- Fig.15 Reactor set up for removal of excess metabolic water by using a reverse osmosis filter outside of the bioreactor (cell retention experiment 4).
- Experimental set-up a: reactor, b: metabolic water, c: R/O membrane, d: cells/nutrients.
- An industrial scale reactor was supplied with H 2 , generated by an electrolyser, and CO 2 , a byproduct of biogas purification. The flow rates of hydrogen and carbon dioxide were adjusted to a 4.1:1 ratio.
- the temperature of the culture was 62.5 °C and the methanation reaction occurred at 10 bar.
- Biocatalyst liquid was removed from the reactor and passed through a R/O membrane to remove the produced metabolic water. The cells and most of the dissolved nutrients were returned to the reactor after passing by the membrane.
- Fig.16 Reactor set up for removal of excess metabolic water by using evaporation (cell retention experiment s). Experimental set-up. a: reactor, b: water vapor, c: condenser, d: metabolic water. Fig. 17. A: photo of the cell morphology of the Methanothermobacter thermautrophicus UC 120910 (ECHOIOO) of experiment 1 from a qualitative control sample derived from cells which were cultured in a growth-phase under no cell retention conditions. As can be seen longer cells predominated.
- ECHOIOO Methanothermobacter thermautrophicus UC 120910
- the inventors of the present invention have set themselves the task to provide a method to convert H 2 and CO 2 into methane by methanogenic microorganisms in a scalable, reliable and continuous production process for methane enriched gas compositions. Therefore, the inventors have tested a new approach to culture methanogenic microorganisms, namely by applying cell retention conditions.
- One method to retain the methanogenic microorganisms was tested by the inventors by means of filtration to remove excess formed metabolic water during the methanation production phase.
- the concept was realized in the form of a ceramic filter unit suitable for microfiltration, which was submerged into the cell culture suspension close to the surface of the cell culture medium inside the reactor (reduced outline of experimental set-up depicted in Fig. 14).
- Ceramic filters were supplied by Katadyn Kunststoff GmbFI and Guangzhou PUREEASY Hi-T ech CO., LTD, with pore sizes of 0.3 mm and 0.1 mm, respectively.
- Filter housings were constructed from A4 stainless steel parts. By using microfiltration, all cells were kept inside the reactor (cell retention).
- the inventors were also interested to test their hypothesis if a given methane productive methanogenic microorganism population under cell retention conditions could still be stably maintained over time under conditions where the supply of the nitrogen source is reduced or even completely stopped.
- the experiment was conducted in a 10 L bioreactor and covered within 1,600 h total running the following different process conditions / phases: a. cell growth.
- phase index a The duration of the growth phase (phase index a) was 165 h when the density of the culture increased up to OD 610 30 followed by further increase to OD 610 80 during a 387 h lasting period of testing different filter materials for the cell retention (phase index b) using a filter with a pore diameter of 0.1 mm or one with a pore diameter of 0.3 mm.
- the OD 610 After chosen a filter with a pore diameter of 0.3 mm for all of the further cell retention phases, the OD 610 reaching a stationary phase, wherein cell density remained very stable during methane production phase without cell retention (duration: 97 h, phase index c, see also in Figures 1 and 2), cell retention condition (duration: 221 h, phase index d), ammonia reduction condition (duration: 440 h, phase index e) and the phase of no ammonia dosing (duration: 216 h, phase index f).
- cell retention condition dueration: 221 h, phase index d
- ammonia reduction condition dueration: 440 h, phase index e
- the phase of no ammonia dosing dueration: 216 h, phase index f
- the flow of the 0.1 mm filter was slow and it needed more than the double time to reach the same volume with the help of the vacuum pump. With both filters, the optical densities and qualitative comparison of the number of cells in the filtrate were the same. The reason of the lower flow of the 0.1 mm filter could also lie in the smaller surface. At longer runtime of the vacuum pump, a general decrease in the flow was also noted. Because of the higher flow, the filter with a pore size of 0.3 mm was used in the following experiments of cell-retention.
- the methane production rate is a measure of process kinetics and often indicated by the volume of methane per volume of cell-suspension and per day (abbreviated as WD in the following).
- WD volume of methane per volume of cell-suspension and per day
- the filter testing period is characterized by changes and adaptations in the experimental setup resulting in a drop with major fluctuations both of the conversion rate and the WD. During these fewer stable conditions, mean conversion rate and WD remain lower than in the subsequent test phases with expected high standard deviation.
- the concentration of ammonia during the decreasing phases shows the dropping NH4 + -concentration from 250 mg NH4+/L initially to 100 mg NH4 + /L at the end of the period with reduced ammonia feeding and 10 mg NH4 + /L at the end of the experiment.
- the pH stabilisation was conducted by addition of NaOH.
- the inventors of the present invention have surprisingly found, that by cultivating methanogenic microorganisms under cell retention conditions according to the present invention it is possible (in this example subsequent a growth phase) to tremendously reduce or even completely stop the supply of the nitrogen source in the methane production phase as still a high and quite stabilized methanation rate was observed in theses phases compared with phases with full ammonium supply over time while maintaining cell culture number (cf. Fig. 1, 2 and 3, especially phase indices e and f compared with d (and additionally c)).
- the experiment 2 was conducted in a 10 L reactor and covered within 6,000 h total running time under the following different process conditions / phases: a. cell growth.
- the results of the experiment 2 are depicted in Figures 4, 5 and 6.
- the duration of the growth phase was 500 h when the density of the culture increased up to OD 40.
- cell density stayed stable within a range of OD 35-50 (cf. Fig. 4 and 5).
- transition phase (cl: 500 h)
- the density of the culture decreased from OD 40 to OD 18.
- the OD was specifically reduced and then within the transition phase a new cell division and cell growth impulse was initiated.
- the OD increased to a level above 60 within 200 h and constantly stayed in a range of OD 60-85 during the following 1.800 h.
- the average CO 2 conversion rate (81 %) was highest in the growth phase.
- the WD (18.7 L/L/d) was lowest and standard deviations were highest during the initial growth phase due to the process- related increase of the flow from 0.05 l to 0.23 l CO 2 per minute during the start-up.
- the concentration of ammonia during the decreasing phase shows the dropping NH4 + -concentration from 373 mg NH4 + /L/d initially to 224 mg NH4 + /L/d at the end of the period with reduced ammonia feeding (cf. Fig. 6).
- the concentration of ammonia in the cell culture medium during the decreasing phase shows the dropping NH4+-concentration from ca. 250 - 200 mg NH4 + /L initially to ca. 100 mg NH4+/L at the end of the period with reduced ammonia feeding at the end of the experiment.
- the WD was not significant different in the different test conditions after the cell growth phase and remained stable between 26.5 and 34.4 L/L/d.
- the VVD 25.9 L/L/d was lowest and standard deviations were highest during the initial growth phase due to the process- related increase of the flow from 0.035 L to 0.3 L CO 2 per minute during the start-up. Similar to the results of the conversation rate and because of some fluctuations.
- Experiment 4 was conducted in a 3,500 L bioreactor and covered within 200 h the following different process conditions/phases: a. methane production without cell retention and culture medium component recycling.
- Reverse Osmosis (R/O) membrane/filter unit For medium recovery a Reverse Osmosis (R/O) membrane/filter unit was used. With this filter/membrane unit metabolic water is removed from the system (permeate) while the cells and minerals which cannot pass the water permeable filter/membrane are accumulated before the filter as retentate and fed back into the reactor with appropriate means, thus allowing to run the reactor system under cell retention conditions.
- R/O Reverse Osmosis
- the OD was stable in a range of OD 34-36.
- the cell density increased to a level above OD 50 within 51 h and constantly stayed in a range between OD 50 and 60 during the following 111 h.
- the reactor was always switched off after about 8 h. After restarting the reactor, the conversion was always in the same range as before the shutdown. After a running time of 83 h, the reactor was operated continuously without interruptions.
- the CO 2 conversion rate was mainly stable in a range of 90-100 %.
- the medium recovery and cell retention had no negative effect on the stability of the process, on the contrary the stability of the process remained unaffected under cell retention conditions and medium recycling.
- An industrial scale reactor with a filling volume of 4500 - 5000 L was used.
- the reactor was supplied with biogas, containing approximately 50% methane and 50% carbon dioxide, from an anaerobic digester. Flydrogen was supplied from a hydrogen tank.
- the content of CO 2 in the biogas was measured using an infrared gas analyzer (IRGA) and the flow rates of biogas and hydrogen were adjusted to achieve a ratio of H 2 : CO 2 that was greater than 4.0.
- IRGA infrared gas analyzer
- the reactor was placed on an industrial-sized scale. When the reactor was empty, the scale was tared. Thus, the weight measured is only the weight of the contents of the reactor.
- the temperature of the reactor headspace was 63 °C and the methanation reaction occurred at atmospheric pressure.
- the range of reactor mass was from 4750 to 4850 kg.
- the mass increased when the biogas flow rate was increased (hours 288-365) as a result of increased CFU and H 2 0 production.
- Water vapor released with the outlet (product) gas flow was the only means of removing the H 2 0 produced by the biocatalyst in the biomethanation reaction.
Abstract
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